## NULL

Supplemental Table 1 - Descriptive statistics of excluded vs included samples with methylation data available from the Fragile and Families and Child Wellbeing study. Samples excluded according to the flow diagram in Main Figure 1

Characteristic M samples Final analysis group, M samples = 14991 Not in final analysis group, M samples = 1861
Prenatal maternal smoking 1,685
Not exposed 1,201 (80%) 141 (76%)
Smoke exposed 298 (20%) 45 (24%)
Age at DNA methylation sample 1,685
Age 15 746 (50%) 110 (59%)
Age 9 753 (50%) 76 (41%)
Polymethylation score: coefficients for sustained smoking from newborn cordblood meta-analysis, w/ cell-type control (mean-centered) 1,685 0.00 (0.23) -0.01 (0.24)
AHHR: cg05575921 1,685 77.1 (5.8) 75.2 (7.4)
Pediatric clock 1,685 10.29 (1.88) 10.52 (1.84)
Global methylation 1,685 47.51 (0.80) 47.42 (0.90)
Immune cell proportion (saliva) 1,685 0.94 (0.13) 0.93 (0.15)
Epithelial cell proportion (saliva) 1,685 0.06 (0.13) 0.07 (0.15)
Ancestry categorization from child principal components of genetic data 1,663
African ancestry 904 (60%) 78 (48%)
European ancestry 235 (16%) 19 (12%)
Hispanic ancestry 360 (24%) 67 (41%)
Unknown 0 22
Child gender 1,685
Boy 740 (49%) 107 (58%)
Girl 759 (51%) 79 (42%)
Maternal prenatal alcohol use 1,681 173 (12%) 20 (11%)
Unknown 0 4
Maternal prenatal any drug use 1,679 73 (4.9%) 11 (6.1%)
Unknown 0 6
Constructed - Poverty ratio - mother's household income/poverty threshold 1,685 2.23 (2.43) 2.35 (2.55)
Any postnatal maternal smoking when child age 1 or 5 1,597
Maternal smoking at age 1 or age 5 590 (39%) 52 (53%)
No maternal smoking at age 1 and 5 909 (61%) 46 (47%)
Unknown 0 88
Maternal/primary care giver smoking in month prior to visit (pks/day) 1,667
Less than pack a day 337 (22%) 42 (25%)
No smoking 1,054 (70%) 115 (68%)
Pack or more a day 108 (7.2%) 11 (6.5%)
Unknown 0 18

1 n (%); Mean (SD)

##    [1] "1000002" "1000005" "1000019" "1000020" "1000024" "1000029" "1000041"
##    [8] "1000042" "1000047" "1000049" "1000052" "1000066" "1000074" "1000084"
##   [15] "1000089" "1000097" "1000099" "1000102" "1000111" "1000129" "1000135"
##   [22] "1000138" "1000186" "1000192" "1000199" "1000201" "1000206" "1000207"
##   [29] "1000223" "1000227" "1000233" "1000236" "1000237" "1000252" "1000254"
##   [36] "1000258" "1000260" "1000271" "1000284" "1000286" "1000291" "1000292"
##   [43] "1000293" "1000294" "1000312" "1000315" "1000316" "1000317" "1000324"
##   [50] "1000326" "1000342" "1000347" "1000361" "1000369" "1000375" "1000378"
##   [57] "1000379" "1000381" "1000389" "1000404" "1000417" "1000418" "1000421"
##   [64] "1000428" "1000429" "1000433" "1000459" "1000475" "1000477" "1000478"
##   [71] "1000484" "1000486" "1000487" "1000488" "1000498" "1000504" "1000507"
##   [78] "1000509" "1000517" "1000538" "1000539" "1000551" "1000552" "1000556"
##   [85] "1000558" "1000560" "1000562" "1000568" "1000580" "1000587" "1000601"
##   [92] "1000603" "1000604" "1000611" "1000622" "1000624" "1000626" "1000627"
##   [99] "1000630" "1000639" "1000653" "1000657" "1000676" "1000680" "1000686"
##  [106] "1000697" "1010204" "1010210" "1010321" "1010387" "1010393" "1010509"
##  [113] "1010771" "1010826" "1010832" "1010848" "1010959" "1011020" "1011153"
##  [120] "1011175" "1011353" "1011369" "1011397" "1011503" "1011842" "1011892"
##  [127] "1012113" "1012535" "1012735" "1012785" "1012907" "1012985" "1013006"
##  [134] "1013206" "1013228" "1013234" "1013345" "1013973" "1013989" "1014016"
##  [141] "1014094" "1014177" "1014199" "1014327" "1014349" "1014511" "1014672"
##  [148] "1014983" "1015004" "1025063" "1025091" "1025130" "1025174" "1025180"
##  [155] "1025263" "1025279" "1025291" "1025318" "1025330" "1025352" "1025479"
##  [162] "1025485" "1025502" "1025530" "1025724" "1025752" "1025796" "1025813"
##  [169] "1025863" "1025885" "1025924" "1026023" "1026039" "1026067" "1026095"
##  [176] "1026106" "1026140" "1026178" "1026190" "1026201" "1026223" "1026251"
##  [183] "1026289" "1026328" "1026340" "1026356" "1026384" "1026417" "1026423"
##  [190] "1026439" "1026451" "1026495" "1026528" "1026590" "1026623" "1026667"
##  [197] "1026706" "1026728" "1026740" "1026756" "1026801" "1026823" "1026839"
##  [204] "1026845" "1026867" "1026895" "1026912" "1026928" "1026934" "1026940"
##  [211] "1026956" "1027005" "1027033" "1027077" "1027138" "1027144" "1027194"
##  [218] "1027205" "1027316" "1027344" "1027388" "1027449" "1027477" "1027499"
##  [225] "1027611" "1027633" "1027649" "1027661" "1027700" "1027738" "1027744"
##  [232] "1028015" "1028021" "1028037" "1028043" "1028093" "1028110" "1028148"
##  [239] "1028160" "1028198" "1028215" "1028221" "1028293" "1028310" "1028354"
##  [246] "1028376" "1028382" "1028415" "1028459" "1028465" "1028471" "1028560"
##  [253] "1028665" "1028710" "1028726" "1028732" "1028754" "1028821" "1028859"
##  [260] "1028893" "1028926" "1028960" "1028982" "1029003" "1029047" "1029097"
##  [267] "1029114" "1029120" "1029142" "1029164" "1029170" "1029203" "1029225"
##  [274] "1029253" "1029275" "1029342" "1029358" "1029847" "1029869" "1029914"
##  [281] "1029920" "1029936" "1029942" "1049021" "1050167" "1050195" "1050240"
##  [288] "1050301" "1050345" "1050478" "1050640" "1050789" "1050917" "1051872"
##  [295] "1051927" "1052232" "1052343" "1052393" "1052454" "1052765" "1052971"
##  [302] "1053070" "1053220" "1053331" "1054135" "1054252" "1054357" "1054496"
##  [309] "1054735" "1065043" "1065093" "1065132" "1065148" "1065459" "1065576"
##  [316] "1065760" "1066081" "1066108" "1066164" "1066231" "1066320" "1066364"
##  [323] "1066508" "1066558" "1066669" "1066675" "1066770" "1066819" "1066920"
##  [330] "1067041" "1067063" "1067091" "1067718" "1067774" "1067835" "1067879"
##  [337] "1067918" "1067924" "1068017" "1068112" "1068156" "1068190" "1068306"
##  [344] "1068467" "1068495" "1069227" "1069455" "1069605" "1069677" "1069699"
##  [351] "1069738" "1069750" "1069877" "1069994" "1088035" "1088041" "1088213"
##  [358] "1088235" "1088479" "1089306" "1089334" "1089401" "1090020" "1090042"
##  [365] "1090070" "1090092" "1090103" "1090119" "1090125" "1090147" "1090303"
##  [372] "1090319" "1090325" "1090347" "1090369" "1090464" "1090547" "1090553"
##  [379] "1090608" "1090614" "1090642" "1090658" "1090703" "1090719" "1090731"
##  [386] "1090775" "1090836" "1090864" "1090892" "1090931" "1090975" "1091191"
##  [393] "1091224" "1091246" "1091424" "1091529" "1091557" "1091624" "1091630"
##  [400] "1091646" "1091846" "1091868" "1092056" "1092101" "1092145" "1092206"
##  [407] "1092212" "1092406" "1092523" "1092545" "1092701" "1092723" "1092739"
##  [414] "1092745" "1092773" "1092828" "1092862" "1092878" "1093022" "1093127"
##  [421] "1093133" "1093149" "1093183" "1093238" "1093250" "1093361" "1093400"
##  [428] "1093438" "1093466" "1093644" "1093672" "1093711" "1093838" "1093850"
##  [435] "1093999" "2000053" "2000561" "2000684" "2001120" "2001463" "2001829"
##  [442] "2002200" "2002886" "2004981" "2005168" "2007344" "2008026" "2008288"
##  [449] "2008330" "2011110" "2011327" "2012559" "2013309" "2016364" "2016623"
##  [456] "2017208" "2017800" "2018469" "2100034" "2100157" "2100322" "2100487"
##  [463] "2100500" "2100898" "2102854" "2102977" "2103196" "2104467" "2105194"
##  [470] "2105356" "2106410" "2200031" "2200141" "2200837" "2201823" "2202644"
##  [477] "2203261" "2203449" "2301176" "2301189" "2303378" "2304199" "2400064"
##  [484] "2400213" "2400307" "2400365" "2400585" "2400828" "2401296" "2401432"
##  [491] "2401678" "2401720" "2401733" "2402017" "2403223" "2403964" "2404280"
##  [498] "2500236" "2500317" "2501170" "2501183" "2501866" "2504041" "2504410"
##  [505] "2506463" "2506670" "2506816" "2506971" "2507116" "2507828" "2507899"
##  [512] "2600026" "2600084" "2600204" "2600576" "2601258" "2601452" "2602312"
##  [519] "2606127" "2606509" "2607634" "2607896" "2609098" "2610281" "2700382"
##  [526] "2700599" "2700861" "2701006" "2701190" "2702131" "2703761" "2703965"
##  [533] "2705170" "2705280" "2705455" "2705675" "2705989" "2706108" "2707709"
##  [540] "2707767" "2707932" "2708164" "2800046" "2800266" "2801896" "2801919"
##  [547] "2802057" "2802277" "2802785" "2804576" "2805355" "2806354" "2806891"
##  [554] "2808284" "2809652" "2900289" "2900331" "2900674" "2902779" "2904230"
##  [561] "3001695" "3008708" "3101029" "3103564" "3104194" "3105614" "3301764"
##  [568] "3302475" "3302734" "3303555" "3304648" "3308291" "3403358" "3506019"
##  [575] "3601024" "4000048" "4000158" "4000187" "4000462" "4000488" "4001704"
##  [582] "4003207" "4005551" "4007575" "4101730" "4103686" "4103880" "4500483"
##  [589] "4502135" "4502850" "4504214" "4504829" "4505394" "4506115" "4506238"
##  [596] "5000186" "5000296" "5001046" "5001428" "5002029" "5002317" "5003222"
##  [603] "5003646" "5004098" "5004975" "5005754" "5100497" "5101166" "5102097"
##  [610] "5102631" "5104189" "5104901" "5105120" "5105285" "5105748" "5108091"
##  [617] "5111046" "5113934" "5116355" "5116672" "5116892" "5202188" "5202447"
##  [624] "5205211" "5205279" "5205318" "5209686" "5212686" "5401167" "5405244"
##  [631] "5500013" "5500026" "5500097" "5500262" "5500314" "5500372" "5500495"
##  [638] "5500602" "5500657" "5502972" "5503405" "5503913" "5503926" "5504019"
##  [645] "5504624" "5504792" "5505199" "5506062" "5600010" "5600049" "5600065"
##  [652] "5600227" "5600256" "5600939" "5602076" "5602351" "5602568" "5602623"
##  [659] "5602940" "5603729" "5603839" "5604061" "5604223" "5604281" "5605248"
##  [666] "6000059" "6000541" "6001032" "6001799" "6100205" "6101495" "6106063"
##  [673] "6106351" "6107431" "6108126" "6109015" "6109060" "6109206" "6200011"
##  [680] "6200312" "6200943" "6201078" "6300157" "6301033" "6301075" "6400264"
##  [687] "6400277" "6401250" "6500290" "6500957" "6501859" "6600022" "6600213"
##  [694] "6600242" "6600268" "6600491" "6600705" "6600857" "6600941" "6601047"
##  [701] "6601953" "6602075" "6602334" "6602745" "6602907" "6700142" "6700757"
##  [708] "6700919" "6701219" "6702577" "6703660" "6800097" "7000281" "7000333"
##  [715] "7001604" "7001659" "7002069" "7002603" "7003055" "7003071" "7003615"
##  [722] "7003644" "7100204" "7101575" "7102079" "7102684" "7103379" "7106211"
##  [729] "7200081" "7200214" "7200285" "7200573" "7201239" "7202775" "7300198"
##  [736] "7300460" "7306710" "7403396" "7500312" "7600115" "7600225" "7601570"
##  [743] "7700154" "7700604" "7701658" "7702013" "7702194" "7703180" "7800588"
##  [750] "7800724" "7800892" "7801189" "7900815" "1000002" "1000005" "1000019"
##  [757] "1000024" "1000029" "1000041" "1000042" "1000047" "1000049" "1000052"
##  [764] "1000066" "1000074" "1000084" "1000089" "1000097" "1000099" "1000111"
##  [771] "1000129" "1000135" "1000138" "1000192" "1000199" "1000201" "1000206"
##  [778] "1000223" "1000227" "1000233" "1000236" "1000237" "1000245" "1000252"
##  [785] "1000254" "1000258" "1000260" "1000284" "1000286" "1000291" "1000293"
##  [792] "1000294" "1000312" "1000315" "1000317" "1000324" "1000326" "1000342"
##  [799] "1000347" "1000361" "1000369" "1000375" "1000378" "1000381" "1000389"
##  [806] "1000404" "1000418" "1000421" "1000428" "1000429" "1000433" "1000459"
##  [813] "1000475" "1000477" "1000478" "1000484" "1000486" "1000487" "1000488"
##  [820] "1000498" "1000504" "1000507" "1000509" "1000517" "1000538" "1000539"
##  [827] "1000551" "1000552" "1000556" "1000558" "1000560" "1000562" "1000568"
##  [834] "1000580" "1000587" "1000601" "1000603" "1000604" "1000611" "1000622"
##  [841] "1000626" "1000627" "1000630" "1000639" "1000653" "1000657" "1000676"
##  [848] "1000686" "1010204" "1010210" "1010321" "1010387" "1010393" "1010509"
##  [855] "1010771" "1010826" "1010832" "1010848" "1010959" "1011020" "1011092"
##  [862] "1011153" "1011175" "1011353" "1011369" "1011397" "1011458" "1011503"
##  [869] "1011842" "1011892" "1012113" "1012513" "1012529" "1012535" "1012785"
##  [876] "1012907" "1012985" "1013006" "1013206" "1013228" "1013234" "1013345"
##  [883] "1013973" "1013989" "1014016" "1014094" "1014177" "1014199" "1014327"
##  [890] "1014349" "1014511" "1014672" "1015004" "1025035" "1025063" "1025091"
##  [897] "1025174" "1025180" "1025263" "1025279" "1025318" "1025330" "1025352"
##  [904] "1025479" "1025485" "1025491" "1025502" "1025524" "1025530" "1025724"
##  [911] "1025752" "1025796" "1025813" "1025835" "1025863" "1025885" "1025924"
##  [918] "1025930" "1025980" "1026023" "1026039" "1026067" "1026095" "1026106"
##  [925] "1026140" "1026178" "1026190" "1026201" "1026223" "1026251" "1026289"
##  [932] "1026328" "1026340" "1026356" "1026362" "1026384" "1026401" "1026417"
##  [939] "1026423" "1026439" "1026451" "1026467" "1026495" "1026528" "1026590"
##  [946] "1026623" "1026645" "1026651" "1026667" "1026706" "1026728" "1026740"
##  [953] "1026756" "1026778" "1026801" "1026823" "1026839" "1026845" "1026867"
##  [960] "1026912" "1026928" "1026940" "1026956" "1026962" "1027005" "1027033"
##  [967] "1027049" "1027077" "1027144" "1027194" "1027205" "1027249" "1027388"
##  [974] "1027449" "1027499" "1027611" "1027633" "1027649" "1027661" "1027700"
##  [981] "1027738" "1028015" "1028021" "1028037" "1028043" "1028110" "1028148"
##  [988] "1028160" "1028182" "1028198" "1028215" "1028221" "1028293" "1028310"
##  [995] "1028354" "1028360" "1028376" "1028382" "1028415" "1028459" "1028465"
## [1002] "1028471" "1028560" "1028665" "1028687" "1028693" "1028710" "1028732"
## [1009] "1028754" "1028821" "1028859" "1028893" "1028960" "1028982" "1029003"
## [1016] "1029047" "1029097" "1029114" "1029120" "1029142" "1029164" "1029170"
## [1023] "1029203" "1029225" "1029253" "1029275" "1029281" "1029342" "1029358"
## [1030] "1029847" "1029869" "1029914" "1029920" "1029936" "1029942" "1049021"
## [1037] "1050167" "1050195" "1050240" "1050301" "1050478" "1050640" "1050789"
## [1044] "1050917" "1051872" "1051927" "1052232" "1052343" "1052393" "1052454"
## [1051] "1052765" "1052971" "1053070" "1053220" "1054135" "1054252" "1054357"
## [1058] "1054735" "1065043" "1065132" "1065148" "1065576" "1065760" "1065776"
## [1065] "1066069" "1066081" "1066108" "1066164" "1066231" "1066320" "1066364"
## [1072] "1066508" "1066536" "1066558" "1066669" "1066675" "1066770" "1066819"
## [1079] "1066920" "1067041" "1067063" "1067091" "1067718" "1067774" "1067835"
## [1086] "1067879" "1067918" "1067924" "1068017" "1068112" "1068190" "1068306"
## [1093] "1068445" "1068467" "1068495" "1069227" "1069455" "1069605" "1069677"
## [1100] "1069699" "1069738" "1069750" "1069877" "1069994" "1088035" "1088041"
## [1107] "1088213" "1088235" "1088479" "1089334" "1089401" "1090020" "1090042"
## [1114] "1090070" "1090092" "1090103" "1090119" "1090147" "1090214" "1090303"
## [1121] "1090319" "1090325" "1090347" "1090369" "1090464" "1090547" "1090553"
## [1128] "1090608" "1090614" "1090642" "1090658" "1090703" "1090719" "1090731"
## [1135] "1090775" "1090836" "1090864" "1090892" "1090931" "1090975" "1091191"
## [1142] "1091224" "1091246" "1091424" "1091529" "1091557" "1091624" "1091630"
## [1149] "1091646" "1091846" "1091868" "1092056" "1092101" "1092145" "1092206"
## [1156] "1092212" "1092406" "1092523" "1092701" "1092723" "1092739" "1092745"
## [1163] "1092773" "1092828" "1092862" "1092878" "1093022" "1093133" "1093149"
## [1170] "1093183" "1093238" "1093250" "1093361" "1093400" "1093438" "1093466"
## [1177] "1093644" "1093672" "1093711" "1093838" "1093850" "1093999" "2000053"
## [1184] "2000561" "2000684" "2001120" "2001463" "2001829" "2002200" "2002886"
## [1191] "2004240" "2004981" "2005168" "2005757" "2007344" "2008026" "2008288"
## [1198] "2008330" "2011110" "2011327" "2012559" "2013309" "2016364" "2016623"
## [1205] "2017208" "2017486" "2017800" "2018469" "2100034" "2100157" "2100322"
## [1212] "2100487" "2100500" "2100898" "2102854" "2102977" "2103196" "2105194"
## [1219] "2105356" "2106410" "2200031" "2200141" "2200837" "2202644" "2203261"
## [1226] "2203449" "2301176" "2301189" "2303378" "2400064" "2400213" "2400307"
## [1233] "2400365" "2400585" "2400828" "2401296" "2401432" "2401678" "2401720"
## [1240] "2401733" "2403223" "2403964" "2500236" "2500317" "2501170" "2501866"
## [1247] "2504410" "2506670" "2506816" "2506971" "2507116" "2507828" "2507899"
## [1254] "2600026" "2600084" "2600204" "2600576" "2601258" "2601452" "2602312"
## [1261] "2606127" "2606509" "2607634" "2607896" "2609098" "2610281" "2700382"
## [1268] "2700599" "2700861" "2701006" "2701190" "2701598" "2702131" "2703761"
## [1275] "2703965" "2705170" "2705280" "2705455" "2705675" "2705989" "2706108"
## [1282] "2707709" "2707767" "2707932" "2708164" "2800046" "2800266" "2801896"
## [1289] "2801919" "2802057" "2802277" "2802785" "2804576" "2806354" "2806891"
## [1296] "2808284" "2809652" "2900137" "2900289" "2900331" "2900593" "2900674"
## [1303] "2902779" "2904230" "2904696" "3001695" "3008708" "3101029" "3103564"
## [1310] "3104194" "3105614" "3301764" "3302475" "3302734" "3303555" "3304648"
## [1317] "3403358" "3506019" "3601024" "4000048" "4000158" "4000462" "4000488"
## [1324] "4000527" "4003207" "4007575" "4101730" "4103686" "4500483" "4502135"
## [1331] "4502850" "4504214" "4504829" "4505394" "4506115" "4506238" "5000296"
## [1338] "5001046" "5001428" "5002029" "5002317" "5003222" "5003646" "5004098"
## [1345] "5004975" "5005754" "5100497" "5102097" "5102631" "5104189" "5104901"
## [1352] "5105120" "5105285" "5105748" "5108091" "5111046" "5113934" "5116355"
## [1359] "5116672" "5116892" "5202188" "5202447" "5205211" "5205279" "5205318"
## [1366] "5212686" "5401167" "5405244" "5500013" "5500026" "5500097" "5500136"
## [1373] "5500262" "5500314" "5500372" "5500495" "5500657" "5500686" "5502972"
## [1380] "5503405" "5503816" "5503913" "5504019" "5504624" "5504637" "5504792"
## [1387] "5505199" "5505872" "5506062" "5506457" "5600010" "5600049" "5600065"
## [1394] "5600227" "5600256" "5600939" "5601695" "5602076" "5602351" "5602568"
## [1401] "5602623" "5602940" "5603729" "5604061" "5604223" "5605248" "6000059"
## [1408] "6000541" "6000619" "6001032" "6001799" "6100205" "6101275" "6101495"
## [1415] "6106351" "6106490" "6106607" "6107431" "6107622" "6108126" "6108223"
## [1422] "6109015" "6109060" "6109206" "6200011" "6200312" "6200943" "6201078"
## [1429] "6300063" "6300157" "6300568" "6301033" "6301075" "6400264" "6400277"
## [1436] "6401250" "6500290" "6500562" "6500957" "6501859" "6600022" "6600213"
## [1443] "6600242" "6600268" "6600491" "6600705" "6600941" "6601047" "6601953"
## [1450] "6602075" "6602334" "6602745" "6700142" "6700919" "6702577" "6702962"
## [1457] "6703660" "6800097" "7000281" "7000333" "7001604" "7001659" "7002069"
## [1464] "7002603" "7003055" "7003071" "7003615" "7003644" "7100204" "7101575"
## [1471] "7102079" "7102684" "7103379" "7106211" "7200081" "7200214" "7200285"
## [1478] "7200573" "7201239" "7300198" "7300460" "7306710" "7403396" "7500312"
## [1485] "7600115" "7600225" "7601570" "7700154" "7700604" "7701658" "7702013"
## [1492] "7702194" "7703180" "7800588" "7800724" "7800892" "7801189" "7900420"
## [1499] "7900815"
## attr(,"label")
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##  "ID"

Supplemental Figure 1 - Pearson correlation of selected DNA methylation summary measures from an anlysis of M=1499 samples and N=809children in the Fragile Families and Child Wellbeing cohort. Lower diaganol shows the Pearson rho correlation coefficient, upper triangle shows ellipses illustrating relationship between variables colored by the magnitude and direction of the correlation.

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##   2

Supplemental Figure 2 - Correlation between age 9 and age 15 visit DNA methylation summary measures among 690 children with age 9 and age 15 visit data in the Fragile Families and Child Wellbeing study. Each point represents one individual, x-axis is the DNA methylation summary measure at age 9 and y-axis is the DNA methylation measure at age 15. Blue lines are simple linear regression lines of best fit, with Pearson rho and associated \(P\) value shown. From top-left: Polymethylation score for prenatal maternal smoking, score created using coefficients from a regression of prenatal maternal smoking and DNA methylation in newborn cord blood, incorporating cell-type control. Polymethylation score for prenatal maternal smoking using coefficients from a regression of prenatal maternal smoking and DNA methylation in newborn cord blood, no cell-type control. Polymethylation score calculated using regression coefficients from a regression of prenatal maternal smoking and DNA methylation in older children’s peripheral blood samples. Pediatric epigenetic clock (years). GRIM epigenetic clock (years). Percent DNA methylation at cg05575921 in AHRR. Percent global DNA methylation (mean across all DNA methylation sites in cleaned probe set). Proportion of epithelial cells calculated from DNA methylation using reference panel of children’s saliva. Proportion of immune cells calculated from DNA methylation using a reference panel of children’s saliva.

Supplemental Figure 3: Distributions of selected methylation summary measures at age 9 and age 15 among 809 children in the Fragile Families and Child Wellbeing study. Captive box plots within violin plots show the distribution of selected DNA methylation measures in samples from age 9 (red) and age 15 (blue). Black lines show lines of best fit. From top-left: Polymethylation score for prenatal maternal smoking, score created using coefficients from a regression of prenatal maternal smoking and DNA methylation in newborn cord blood, incorporating cell-type control. Polymethylation score for prenatal maternal smoking using coefficients from a regression of prenatal maternal smoking and DNA methylation in newborn cord blood, no cell-type control. Polymethylation score calculated using regression coefficients from a regression of prenatal maternal smoking and DNA methylation in older children’s peripheral blood samples. Pediatric epigenetic clock (years). GRIM epigenetic clock (years). Percent DNA methylation at cg05575921 in AHRR. Percent global DNA methylation (mean across all DNA methylation sites in cleaned probe set). Proportion of epithelial cells calculated from DNA methylation using reference panel of children’s saliva. Proportion of immune cells calculated from DNA methylation using a reference panel of children’s saliva.

Characteristic Age 9 Age 15
N Not exposed, N = 5981 Smoke exposed, N = 1551 p-value2 N Not exposed, N = 6031 Smoke exposed, N = 1431 p-value2
Child characteristics
Polymethylation score for smoke exposure 753 -0.04 (0.22) 0.08 (0.22) <0.001 746 -0.01 (0.22) 0.11 (0.23) <0.001
AHRR gene: percent cg05575921 methylation 753 77.8 (5.2) 76.8 (5.6) 0.047 746 77 (6) 76 (6) 0.2
Pediatric epigenetic clock (years) 753 8.98 (0.75) 8.94 (0.83) 0.6 746 11.62 (1.75) 11.59 (1.70) 0.9
Percent global DNA methylation 753 47.51 (0.74) 47.59 (0.78) 0.3 746 47.48 (0.85) 47.58 (0.83) 0.2
Immune cell proportion (saliva) 753 0.96 (0.10) 0.96 (0.08) 0.4 746 0.92 (0.15) 0.92 (0.14) >0.9
Epithelial cell proportion (saliva) 753 0.04 (0.10) 0.04 (0.08) 0.4 746 0.08 (0.15) 0.08 (0.14) >0.9
Ancestry categorization from child principal components of genetic data 753 0.001 746 0.001
African ancestry 355 (59%) 97 (63%) 364 (60%) 88 (62%)
European ancestry 82 (14%) 35 (23%) 84 (14%) 34 (24%)
Hispanic ancestry 161 (27%) 23 (15%) 155 (26%) 21 (15%)
Child gender 753 0.8 746 0.9
Boy 300 (50%) 76 (49%) 295 (49%) 69 (48%)
Girl 298 (50%) 79 (51%) 308 (51%) 74 (52%)
Maternal characteristics
Maternal prenatal alcohol use 753 44 (7.4%) 47 (30%) <0.001 746 40 (6.6%) 42 (29%) <0.001
Maternal prenatal any drug use 753 10 (1.7%) 26 (17%) <0.001 746 9 (1.5%) 28 (20%) <0.001
Maternal income to poverty threshold (at baseline) 753 2.41 (2.57) 1.52 (1.56) <0.001 746 2.41 (2.58) 1.49 (1.57) <0.001
Postnatal maternal smoking at ages 1 or 5 753 <0.001 746 <0.001
No maternal smoking at age 1 and 5 447 (75%) 7 (4.5%) 448 (74%) 7 (4.9%)
Maternal smoking at age 1 or age 5 151 (25%) 148 (95%) 155 (26%) 136 (95%)
Postnatal primary caregiver smoking in past month prior to visit 753 <0.001 746 <0.001
No smoking 476 (80%) 23 (15%) 511 (85%) 44 (31%)
Less than pack a day 95 (16%) 85 (55%) 80 (13%) 77 (54%)
Pack or more a day 27 (4.5%) 47 (30%) 12 (2.0%) 22 (15%)

1 Mean (SD); n (%)

2 Welch Two Sample t-test; Pearson's Chi-squared test

Supplemental Table 2: Bivariate associations between prenatal maternal smoking and additional DNA methylation summary measures among a diverse sample of 809 children in the Fragile Families and Child Wellbeing study. Percent DNA methylation shown calculated across all DNA methylation sites available (global DNA methylation) and stratified by genomic region (Island, Open Sea, Shelf, Shore). Polymethylation score for smoke exposure from main analysis and from sensitivity analyses where scores were calculated using alternative available regression coefficients.

Characteristic Age 9 Age 15
N Not exposed, N = 5981 Smoke exposed, N = 1551 p-value2 N Not exposed, N = 6031 Smoke exposed, N = 1431 p-value2
Percent global DNA methylation 753 47.51 (0.74) 47.59 (0.78) 0.3 746 47.48 (0.85) 47.58 (0.83) 0.2
Island methylation 753 18.96 (0.61) 19.06 (0.59) 0.057 746 19.05 (0.61) 19.14 (0.56) 0.10
Open Sea methylation 753 70.36 (1.05) 70.41 (1.11) 0.6 746 70.23 (1.23) 70.35 (1.22) 0.3
Shelf methylation 753 75.33 (1.04) 75.35 (1.10) 0.9 746 75.19 (1.23) 75.30 (1.21) 0.4
Shore methylation 753 44.05 (0.82) 44.16 (0.86) 0.2 746 44.03 (0.94) 44.15 (0.90) 0.2
Pediatric epigenetic clock (years) 753 8.98 (0.75) 8.94 (0.83) 0.6 746 11.62 (1.75) 11.59 (1.70) 0.9
GRIM epigenetic clock (years) 753 24.7 (3.5) 24.5 (3.2) 0.6 746 30.4 (4.0) 30.2 (4.2) 0.6
GRIM pack/yrs component 753 -5.7 (4.7) -4.7 (5.0) 0.033 746 -4.0 (5.3) -3.6 (5.1) 0.4
Epithelial cell proportion (saliva) 753 0.04 (0.10) 0.04 (0.08) 0.4 746 0.08 (0.15) 0.08 (0.14) >0.9
Immune cell proportion (saliva) 753 0.96 (0.10) 0.96 (0.08) 0.4 746 0.92 (0.15) 0.92 (0.14) >0.9
Polymethylation score for smoke exposure 753 -0.04 (0.22) 0.08 (0.22) <0.001 746 -0.01 (0.22) 0.11 (0.23) <0.001
Polymethylation score: coefficients for sustained smoking from newborn cordblood meta-analysis (mean-centered) 753 -0.06 (0.29) 0.05 (0.27) <0.001 746 0.01 (0.31) 0.12 (0.31) <0.001
Polymethylation score: coeffcients for sustained smoking from older children peripheral blood meta-analysis (mean-centered) 753 -0.02 (0.23) 0.09 (0.25) <0.001 746 -0.02 (0.27) 0.07 (0.28) <0.001
Polymethylation score: coefficients for any smoking from newborn cordblood meta-analysis (mean-centered) 753 -0.04 (0.21) 0.04 (0.19) <0.001 746 0.01 (0.22) 0.09 (0.22) <0.001
AHRR gene: percent cg05575921 methylation 753 77.8 (5.2) 76.8 (5.6) 0.047 746 77 (6) 76 (6) 0.2
MYO1G: cg04180046 753 35 (7) 39 (8) <0.001 746 35 (8) 38 (8) <0.001
CYP1A1: cg05549655 753 18.7 (5.1) 20.9 (5.5) <0.001 746 18.2 (5.2) 20.5 (5.1) <0.001
GFI1: cg14179389 753 5.67 (2.81) 4.89 (2.62) 0.001 746 5.73 (2.87) 4.73 (1.79) <0.001
MYO1G: cg22132788 753 69 (10) 73 (9) <0.001 746 67 (11) 70 (12) 0.006

1 Mean (SD)

2 Welch Two Sample t-test

Supplemental Figure 4: Sensitivity analyses for controlling for different variable sets in multivariable analyses of prenatal maternal smoking and DNA methylation summary measures. Base model: Child sex, maternal income to poverty ratio, immune cell proportion, plate, 1st two principal components from PCA on child genetic data, child age. Prenatal exposures model: base model + yes/no any prenatal maternal alcohol use, yes/no any prenatal other drug use. Secondhand smoking models: prenatal exposures model + yes/no any postnatal maternal/primary care giver smoking at age 1 or 5, pk/day in month prior to primary caregiver/maternal interview at age 9 or 15. Surrogate variable: including all surrogate variables

Supplemental Figure 5 DNA methylation summary measures across combinations of exposure to prenatal and postnatal maternal smoking. Sample size is shown at the bottom of violin plots with captured boxplots. POlymethylation score shown in top facets, percent DNA methylation at cg05575921 in AHRR gene shown in bottom facets, age 9 samples shown in left facets, age 15 shown in right facets

Supplemental Figure 6: Heatmap of P values from Pearson correlations between known covariates and surrogate variables from surrogate variable models

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##   2

Supplemental Table 3: Coefficients for multivariable models of prenatal maternal smoke exposure regressed on selected DNA methylation summary variables (outcomes) among children in the Fragile Families and Child Wellbeing study using surrogate variables

Characteristic Global methylation Pediatric clock Sustained smoking polymethylation score (newborns, cell-type controlled) AHRR: cg05575921
N Beta 95% CI1 p-value N Beta 95% CI1 p-value N Beta 95% CI1 p-value N Beta 95% CI1 p-value
African ancestry
Prenatal maternal smoke exposed 904 0.01 0.01, 0.02 <0.001 904 -0.21 -0.40, -0.02 0.032 904 0.41 0.31, 0.51 <0.001 904 -1.0 -1.5, -0.55 <0.001
European ancestry
Prenatal maternal smoke exposed 235 0.01 0.01, 0.02 <0.001 235 0.03 -0.18, 0.25 0.8 235 0.60 0.39, 0.81 <0.001 235 -1.2 -1.9, -0.41 0.003
Hispanic ancestry
Prenatal maternal smoke exposed 360 0.00 -0.01, 0.01 0.4 360 -0.05 -0.38, 0.28 0.8 360 0.36 0.16, 0.56 <0.001 360 -0.55 -1.5, 0.39 0.2
Unstratified
Prenatal maternal smoke exposed 1499 0.01 0.01, 0.02 <0.001 1499 -0.17 -0.31, -0.03 0.020 1499 0.42 0.34, 0.50 <0.001 1499 -0.92 -1.3, -0.54 <0.001

1 CI = Confidence Interval

Supplemental Table 4: Associations between prenatal maternal smoking and DNA methylation summary measures in multivariable linear models stratified by age or in multivariable linear mixed effect models among 809 US children. All models controlled for: first two principal components of child genetic ancestry (recalculated within ancestry strata for stratified models), child sex, maternal income-to-poverty ratio at birth, immune cell proportion estimated from methylation data, child age

Characteristic Global methylation Pediatric clock Sustained smoking polymethylation score (newborns, cell-type controlled) AHRR: cg05575921
N Beta 95% CI1 p-value N Beta 95% CI1 p-value N Beta 95% CI1 p-value N Beta 95% CI1 p-value
Age 9 models
Prenatal maternal smoke exposed 753 0.02 -0.08, 0.12 0.6 753 -0.07 -0.18, 0.04 0.2 753 0.52 0.36, 0.67 <0.001 753 -1.0 -1.7, -0.35 0.003
Age 15 models
Prenatal maternal smoke exposed 746 0.09 -0.02, 0.20 0.12 746 -0.04 -0.24, 0.16 0.7 746 0.46 0.30, 0.62 <0.001 746 -0.80 -1.6, -0.02 0.043
Longitudinal models
Prenatal maternal smoke exposed 1499 0.05 -0.03, 0.14 0.2 1499 -0.02 -0.16, 0.11 0.7 1499 0.48 0.34, 0.62 <0.001 1499 -0.92 -1.5, -0.35 0.002

1 CI = Confidence Interval

Supplemental Table 5: Associations between prenatal maternal smoking and DNA methylation summary measures in multivariable models among 809 US children by ancestry using linear mixed effect models. All models controlled for: first two principal components of child genetic ancestry (recalculated within ancestry strata for stratified models), child sex, maternal income-to-poverty ratio at birth, immune cell proportion estimated from methylation data, child age

Characteristic Global methylation Pediatric clock Sustained smoking polymethylation score (newborns, cell-type controlled) AHRR: cg05575921
N Beta 95% CI1 p-value N Beta 95% CI1 p-value N Beta 95% CI1 p-value N Beta 95% CI1 p-value
African ancestry
Prenatal maternal smoke exposed 904 0.05 -0.05, 0.16 0.3 904 0.05 -0.13, 0.22 0.6 904 0.53 0.34, 0.71 <0.001 904 -1.2 -1.9, -0.46 0.001
European ancestry
Prenatal maternal smoke exposed 235 -0.07 -0.27, 0.12 0.5 235 0.01 -0.24, 0.25 >0.9 235 0.41 0.04, 0.77 0.030 235 -1.2 -2.5, 0.01 0.052
Hispanic ancestry
Prenatal maternal smoke exposed 360 0.11 -0.10, 0.31 0.3 360 -0.28 -0.63, 0.06 0.11 360 0.25 -0.08, 0.58 0.13 360 0.30 -1.1, 1.7 0.7
Unstratified
Prenatal maternal smoke exposed 1499 0.05 -0.03, 0.14 0.2 1499 -0.02 -0.16, 0.11 0.7 1499 0.48 0.34, 0.62 <0.001 1499 -0.92 -1.5, -0.35 0.002

1 CI = Confidence Interval

Supplemental Figure 7: Associations between prenatal maternal smoking and polymethylation scores calculated using alternative coefficient sets among n n_total children in the Fragile Families and Child Wellbeing study. Base model: Child sex, maternal income to poverty ratio, immune cell proportion, plate, 1st two principal components from PCA on child genetic data, child age. Prenatal exposures model: base model + yes/no any prenatal maternal alcohol use, yes/no any prenatal other drug use. Secondhand smoking models: prenatal exposures model + yes/no any postnatal maternal/primary care giver smoking at age 1 or 5, pk/day in month prior to primary caregiver/maternal interview at age 9 or 15. Surrogate variable: including all surrogate variables

Supplemental Figure 8 - Associations between prenatal maternal smoking and other a priori CpG sites selected from previous meta-analyses among 809 children in the Fragile Families and Child Wellbeing study

Supplemental Table 6: Area under the curve for models shown in Main Figure 4 for classifying prenatal maternal smoke exposure, with P value when comparing to base model only or to model using polymethylation score + base model. Base model: Child sex, maternal income to poverty ratio, immune cell proportion, plate, 1st two principal components from PCA on child genetic data, child age.

Age Model AUC P value vs base model P value vs base model + polymethylation score
Age 9 Global methylation 0.53 0.0000 0.0000
Age 9 Sustained smoking polymethylation score (newborns, cell-type controlled) 0.66 0.0514 0.0000
Age 9 AHRR: cg05575921 0.55 0.0000 0.0000
Age 9 Base model+No methylation 0.73 1.0000 0.0003
Age 9 Base model+Global methylation 0.73 0.3522 0.0004
Age 9 Base model+Sustained smoking polymethylation score (newborns, cell-type controlled) 0.77 0.0003 1.0000
Age 9 Base model+AHRR: cg05575921 0.74 0.1639 0.0256
Age 15 Global methylation 0.54 0.0000 0.0000
Age 15 Sustained smoking polymethylation score (newborns, cell-type controlled) 0.65 0.0274 0.0000
Age 15 AHRR: cg05575921 0.53 0.0000 0.0000
Age 15 Base model+No methylation 0.72 1.0000 0.0008
Age 15 Base model+Global methylation 0.72 0.3976 0.0008
Age 15 Base model+Sustained smoking polymethylation score (newborns, cell-type controlled) 0.77 0.0008 1.0000
Age 15 Base model+AHRR: cg05575921 0.73 0.4670 0.0056

Supplemental Table 7: Area under the curve when using different coefficient sets to construct polymethylation scores for predicting prenatal maternal smoke exposure. With P value when comparing to base model only or to model using polymethylation score + base model. Base model: Child sex, maternal income to poverty ratio, immune cell proportion, plate, 1st two principal components from PCA on child genetic data, child age.

Age Model AUC P value vs base model P value vs base model + polymethylation score with cell-type control
Age 9 Any smoking polymethylation score (newborns) 0.6197 0.0010 0.0000
Age 9 Sustained smoking polymethylation score (newborns, cell-type controlled) 0.6650 0.0514 0.0000
Age 9 Sustained smoking polymethylation score (newborns) 0.6178 0.0008 0.0000
Age 9 Sustained smoking polymethylation score (older children) 0.6333 0.0037 0.0000
Age 9 Base model+No methylation 0.7260 1.0000 0.0003
Age 9 Base model+Any smoking polymethylation score (newborns) 0.7597 0.0033 0.0117
Age 9 Base model+Sustained smoking polymethylation score (newborns, cell-type controlled) 0.7741 0.0003 1.0000
Age 9 Base model+Sustained smoking polymethylation score (newborns) 0.7596 0.0032 0.0120
Age 9 Base model+Sustained smoking polymethylation score (older children) 0.7676 0.0009 0.4602
Age 15 Any smoking polymethylation score (newborns) 0.6062 0.0007 0.0000
Age 15 Sustained smoking polymethylation score (newborns, cell-type controlled) 0.6471 0.0274 0.0000
Age 15 Sustained smoking polymethylation score (newborns) 0.6054 0.0006 0.0000
Age 15 Sustained smoking polymethylation score (older children) 0.5921 0.0002 0.0000
Age 15 Base model+No methylation 0.7213 1.0000 0.0008
Age 15 Base model+Any smoking polymethylation score (newborns) 0.7565 0.0012 0.0966
Age 15 Base model+Sustained smoking polymethylation score (newborns, cell-type controlled) 0.7669 0.0008 1.0000
Age 15 Base model+Sustained smoking polymethylation score (newborns) 0.7566 0.0012 0.1073
Age 15 Base model+Sustained smoking polymethylation score (older children) 0.7533 0.0078 0.1022